1. Field of Invention
The present invention relates to a mass spectrometer in general and in particular to a miniaturized sample scanning mass spectrometer.
2. Description of Related Art
Mass spectrometers are instruments that are used to determine the chemical composition of substances and the structures of molecules. In general they consist of an ion source where neutral molecules are ionized, a mass analyzer where ions are separated according to their mass/charge ratio, and a detector. Mass analyzers come in a variety of types, including magnetic field (B) instruments, combined electrical and magnetic field or double-focusing instruments (EB or BE), quadrupole electric field (Q) instruments, and time-of-flight (TOF) instruments. In addition, two or more analyzers may be combined in a single instrument to produce tandem (MS/MS) mass spectrometers. These include triple analyzers (EBE), four sector mass spectrometers (EBEB or BEEB), triple quadrupoles (QqQ) and hybrids (such as the EBqQ).
In tandem mass spectrometers, the first mass analyzer is generally used to select a precursor ion from among the ions normally observed in a mass spectrum. Fragmentation is then induced in a region located between the mass analyzers, and the second mass analyzer is used to provide a mass spectrum of the product ions. Tandem mass spectrometers may be utilized for ion structure studies by establishing the relationship between a series of molecular and fragment precursor ions and their products.
Alternatively, they are now commonly used to determine the structures of biological molecules in complex mixtures that are not completely fractionated by chromatographic methods. These may include mixtures of (for example) peptides, glycopeptides or glycolipids. In the case of peptides, fragmentation produces information on the amino acid sequence.
One type of mass spectrometers is time-of-flight (TOF) mass spectrometers. The simplest version of a time-of-flight mass spectrometer, illustrated in FIG. 1 (Cotter, Robert J., Time-of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research, American Chemical Society, Washington, D.C., 1997), the entire contents of which is hereby incorporated by reference, consists of a short source region 10, a longer field-free drift region 12 and a detector 14. Ions are formed and accelerated to their final kinetic energies in the short source region 10 by an electric field defined by voltages on a backing plate 16 and drawout grid 18. The longer field-free drift region 12 is bounded by drawout grid 18 and an exit grid 20.
In the most common configuration, the drawout grid 18 and exit grid 20 (and therefore the entire drift length) are at ground potential, the voltage on the backing plate 16 is V, and the ions are accelerated in the source region to an energy: mv2/2=z eV, where m is the mass of the ion, v is its velocity, z the number of charges, and e is the charge on an electron. The ions then pass through the drift region 12 and their (approximate) flight time(s) is given by the formula:t=[(m/z)/2 eV]1/2D  (I)which shows a square root dependence upon mass. Typically, the length s of source region 10 is of the order of 0.5 cm, while drift lengths (D) ranges from 15 cm to 8 meters. Accelerating voltages (V) can range from a few hundred volts to 30 kV, and flight time are of the order of 5 to 100 microseconds. Generally, the accelerating voltage is selected to be relatively high in order to minimize the effects on mass resolution arising from initial kinetic energies and to enable the detection of large ions. For example, the accelerating voltage of 20 KV (as illustrated, for example, in FIG. 1) has been found to be sufficient for detection of masses in excess of 300 kDaltons.
In recent years, the development of an ionization technique for mass spectrometers known as matrix-assisted laser desorption ionization (MALDI) has generated considerable interest in the use of time-of-flight mass spectrometers and in improvement of their performance. MALDI is particularly effective in ionizing large molecules (e.g. peptides and proteins, carbohydrates, glycolipids, glycoproteins, and oligonucleotides) as well as other polymers. The TOF mass spectrometer provides an advantage for MALDI analysis by simultaneously recording ions over a broad mass range, which is the so called multichannel advantage. In MALDI method of ionization, biomolecules to be analyzed are recrystallized in a solid matrix (e.g., sinnipinic acid, 3-hydroxy picolinic acid, etc.) of a low mass chromophore that its is strongly absorbing in the wavelength region of the pulsed laser used to initiate ionization. Following absorption of the laser radiation by the matrix, ionization of the analyte molecules occurs as a result of desorption and subsequent charge exchange processes. In TOF instruments, all ion optical elements and the detector are enclosed within a vacuum chamber to ensure that ions, once formed, reach the detector without collisions with the background gas.
A number of techniques have been developed to improve the mass resolution of time-of-flight mass spectrometers. Mass resolution is reduced by the initial distributions in the velocity and position of the ions when they are formed. The simplest of the techniques used to improve resolution is the incorporation of a two stage extraction system to provide space focusing at the detector for an instrument with a long drift length, and a second order space-focusing for an optimal drift length (Cotter, R. J., Time-of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research, American Chemical Society, Washington, D.C. 1997, Boesl, U., Weinkauf, R., Schlag, E. W., Int. J. Mass Spectrom. Ion Processes 112 (1992) 121–166). Pulsed extraction and time-delayed extraction techniques have been used to address both space and energy (velocity) focusing (Wiley, W. C., McLaren, I. H., Rev. Sci. Instrumen. 26 (1955) 1150–1157), including the correlated space/velocity distributions proposed for MALDI (Colby, S. M., King, T. B., Reilley, J. P., Rapid Commun. Mass Spectrom. 8 (1994) 865).
Other improvements introduced into time-of-flight spectrometers include miniaturization. An example of a miniaturized TOF mass analyzer is shown in FIG. 2 (Cotter, R. J., The New Time-of-Flight Mass Spectrometery, Anal. Chem 71 (1999) 445A–451A). In mass analyzer instrument 22, samples are presented as a 10×10 array of sample spots (not shown), mounted on a movable XY stage 24. The length of the ion source, i.e. the distance between the surface of movable stage 24 and grid 26, is about 1 inch (2.54 cm) and the length of the drift region 28 is 3 inches (7.6 cm). In mass spectrometer 22 the sample stage 24 and grid 30 are initially at a potential of 6.45 kV. The grid 26 is connected to the drift tube liner and is held at a potential between −1.9 and 12.2 kV of the first channel plate of a gridless Hamamatsu dual channel plate detector 32 (a dual channel plate detector is used to increase the gain of the signal detected). This arrangement eliminates any post acceleration of the ions into the detector. Moreover, this arrangement enables the amplified current pulse from the detector to be taken near ground potential. The drift tube liner is a half inch thin-walled tubing that insures an equipotential (field free) region across the drift length. The sample surface can be, for example, pulsed from 6.45 kV to 9.75 kV, i.e. using a 3.3 kV pulse (as illustrated in FIG. 2). The delay time for pulsing is adjusted to provide best focusing to the front channel plate for a given mass, i.e. maximum mass resolution.
Examples of mass spectra from this instrument are shown in FIGS. 3A and 3B. The mass spectrum in FIG. 3A records peptide biomarkers from Bacillus globigii spores. Although this 3-inch mass analyzer is considerably smaller than the one meter or larger mass analyzers common in commercial instruments, the mass range is limited only by the kinetic energy of the ions at the time they reach the detector. In this case, depending on the drift region bias voltage, the kinetic energy is from 11.6 keV to 11.95 keV. This is sufficient to record ions as large as 66 kdalton molecular ion of bovine serum albumin as shown in the mass spectrum of FIG. 3B.
Most commercial MALDI time-of-flight spectrometers now provide analysis of multiple samples loaded at the same time on a sample holder into the vacuum system. The multiple samples on the sample holder include, for example, “slides” which are one dimensional arrangements from 8 to 30 sample spots, large format two-dimensional arrays using 96 or 384 samples similar to those used in microtiter plates (Vestal M. L., Mass Spectrometer System and Method for Matrix-Assisted Laser Desorption Measurements, U.S. Pat. No. RE37485E, Dec. 25, 2001, U.S. Pat. No. 5,498,545, Mar. 12, 1996), and higher density microarrays or “samples on a chip.”
An example of a two dimensional array sample holder is shown in FIG. 4. Two dimensional array sample holder 40 holds a plurality of samples 42. The plurality of samples are in this case a two dimensional array of samples. The samples on the sample holder are loaded into a vacuum system for mass analysis. Once samples are loaded into the vacuum system of the mass spectrometer, the conventional way to select a sample for analysis is by moving the sample array. Two dimensional arrays of samples on the sample holder 40 are conventionaly mounted on an XY translational stage 44, controlled either manually or by a computer-data system, to bring each sample into the focal point of a laser beam (the ionizer) and the ion extraction optics (not shown in this Figure) which are for example aligned relative to axis AA′ perpendicular to the plane of the sample holder. In addition to sample selection, movement of the sample stage also enables the selection of an area on each sample where the ion signal is more intense. Sample holder 40 is positioned on XY translational stage 44 comprising positioning stage represented by arrow 44X in the X direction and positioning stage 44Y represented by arrow Y in the Y direction.
In this common arrangement, the laser beam and optics, and the ion extraction optics, flight lengths and detectors and other portions of the mass analyzer are stationary within the instruments. It is also common that the sample surface or stage be biased at some high electrical potential. The high potential is used to define the ion kinetic energy. The flight tube (not shown) is, hence, biased near ground potential. The signal from the ion detector is taken either at or close to ground through a 50 ohm output.
In this arrangement, the volume required to accommodate a two dimensional sample array is considerable, and defines both the overall instrument dimensions as well as the capacity of the mechanical and turbomolecular pumping system. Indeed, the sample plate must be moved across an area 46, defining the footprint of the movement of the XY stage, equal to more than 4 times the area of the sample plate or sample holder 40 in order to accommodate and analyze each of the samples 42 across the entire sample array 40. Furthermore, since the sample stage is biased at some high voltage, an additional space 48 (in the X direction) and 49 (in the Y direction) is reserved between the walls of the vacuum chamber and the maximum extension of the XY stage such that the stage would not come in contact with the wall of the vacuum chamber held at a ground potential.
For example, for a 127×86 mm sample array format, the total area needed for the movement of the sample plate alone would be at least 25.4×17.2 cm. In addition, the XY stage and its associated drive mechanisms, some or all of which may be at high voltage, contribute to the depth of a large volume within the mass spectrometer needed to accommodate the entire sample handling system.